ApoA-I is the major protein component of HDL that is required both for the biogenesis and the functions of HDL. ApoA-I also contributes to the overall lipid homeostasis in the circulation and to atheroprotection. Biogenesis, maturation, remodeling and catabolism of HDL is a complex biological process that requires functional interactions of apoA-I with several proteins, including ABCA1, LCAT, SR-BI, PLTP and others. It is our hypothesis, based on our previous work, that subtle changes in the structure of apoA-I may affect not only the biogenesis and the atheroprotective function of HDL, but may also affect its interaction with other proteins of the HDL pathway and thus may cause dyslipidemia. The high resolution 3D structure of apoA-I that was acquired recently, combined with new functional in vivo data we have obtained, allows a systematic approach to map the domains and residues of apoA-I that confer its diverse biological functions that ultimately lead to atheroprotection. Our specific aims are: 1) To determine the effect of natural or bioengineered apoA-I mutations on the activation of LCAT and their impact on HDL levels, HDL subpopulations and HDL functions. The studies will involve adenovirus-mediated gene transfer of apoA-I mutants in apoA-I-deficient mice and in vitro assays that reflect different functions of apoA-I and HDL. 2) To determine by gene transfer and in vitro studies the crucial domains and residues of apoA-I that affect its functional interaction with ABCA1 and PLTP, two proteins involved in the biogenesis and remodeling of HDL, or may cause dyslipidemia when mutated. The in vitro and in vivo functions of apoA-I mutants of Aims 1&2 will be correlated with their physicochemical properties (Aim 3) and the apoA-I structure. 3) To determine by physicochemical methods how alterations in specific domains and residues affect the conformation and the structure of apoA-I due to changes in intra- or intermolecular interaction in solution or when bound to lipids and how the observed changes correlate with alterations in the apoA-I functions that will be studied in Aims 1,2&4. 4) To determine the contribution of apoA-I mutations that affect the biogenesis and maturation of HDL or cause dyslipidemia to the development of atherosclerosis using transgenic mice. Epidemiological and genetic data, combined with transgenic experiments, suggest that increased apoA-I and HDL levels protect from atherosclerosis. In contrast, low apoA-I and HDL levels predispose humans to coronary artery disease, a leading cause of mortality worldwide. This proposal focuses on the basic molecular mechanisms which determine the biological functions of apoA-I that are relevant to the development of CAD. Understanding these mechanisms may lead to new pharmacological approaches to increase HDL levels and thus decrease the risk for cardiovascular disease. PUBLIC HEALTH RELEVANCE: ApoA-I is required for the synthesis of high density lipoprotein, which represents the good cholesterol in our bloodstream. In the proposed project we will investigate how changes in apoA-I affect the synthesis as well as the atheroprotective functions of HDL. This information is important in the design of new pharmaceuticals to increase HDL levels while preserving the atheroprotective functions of HDL and thus decrease the risk for cardiovascular disease.